Enhanced activation of natural killer cells using an NK cell activator and a hydrogen peroxide scavenger or inhibitor

ABSTRACT

A method for providing activated natural killer (NK) cells comprising the steps of administering to a population of cells which includes lymphocytes and monocytes, an effective amount of an NK cell activating cytokine or a NK cell activating flavonoid, wherein said NK cell activating cytokine is not IL-2 or IFN-α; and administering a compound effective to inhibit the production or release of hydrogen peroxide selected from the group consisting of histamine, other H 2  receptor agonists, and serotonin.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of application Ser. No.09/516,641, filed on Mar. 1, 2000, which is a continuation ofapplication Ser. No. 08/681,108, filed Jul. 22, 1996, now U.S. Pat. No.6,071,509, which is a continuation of application Ser. No. 08/287,200,filed Aug. 8, 1994, now abandoned.

This application is related to U.S. application Ser. No. 07/843,052,filed Mar. 2, 1992. FIELD OF THE INVENTION

[0002] The present invention relates generally to methods for theenhanced activation of natural killer (NK) cells, useful for example, inthe treatment of cancer and viral infection. More specifically, thepresent invention relates to the activation of NK cells using acombination of a natural killer cell activator and a hydrogen peroxideinhibiting compound or scavenger. It also relates to the prevention ofinactivation of NK cells.

BACKGROUND OF THE INVENTION DESCRIPTION OF THE RELATED ART

[0003] Natural killer (NK) cells are a subset of spontaneously cytotoxiclymphocytes that lytically destroy tumor cells without apparent antigenspecificity or restriction by histocompatibility molecules. Lymphokinesare lymphocyte-derived peptides that modulate immunologic andinflammatory responses by regulating the activity, growth anddifferentiation of a wide variety of leukocyte and nonleukocyte targetcells. Similar factors produced by a variety of cell types, togetherwith lymphokines, are known as cytokines. Several cytokines are known tostimulate proliferation of NK cells and to enhance their cytotoxicactivity.

[0004] Interleukin-2 (IL-2), formerly T-cell growth factor (TCGF), is aT-cell-derived cytokine. Since 1985, IL-2 has been used in the treatmentof human neoplasia, mainly in patients with metastasizing solid tumors,such as malignant melanoma and renal cell carcinoma (Rosenberg et al.,N. Engl. J. Med. 316:889-897 (1987); Bukowski et al., J. Clin. Oncol.7:477-485 (1989)), but more recently also in acute myelogenous leukemia(AML) (Foa et al., Br. J. Haematol. 77:491-496.3 (1991)). In the initialstudies, IL-2 was administered together with autologous lymphocytes thathad been treated with IL-2 in vitro, but in recent years IL-2 has morefrequently been administered as a single agent.

[0005] The high expectations for the treatment of human cancer usingIL-2 were based on the findings that treatment with IL-2 can induce theregression of established tumors in several animal tumor models in vivo(Rosenberg et al., J. Exp. Med. 161:1169-1188 (1985); Lotre andRosenberg, in Interleukin-2, K. A. Smith, ed., Academic Press, SanDiego, pp. 237-294 (1988)). The mechanism underlying this anti-tumoreffect of IL-2 has been much debated, but accumulating evidence pointsto the anti-tumor effector cell as the natural killer (NK)-cell.Depletion of NK cells from experimental animals eliminates theanti-tumor effect of IL-2 in many experimental models for tumor growthand metastasis (Mule et al., J. Immunol. 139:285 (1987)). Further, theonly subset of resting human peripheral blood lymphocytes that carrytransducing receptors for IL-2 (IL-2R) on the cell surface are NK cells(Caliguri et al., J. Clin. Invest. 91:123-132 (1993)).

[0006] IL-2 activates many NK-cell functions, including baseline or“natural” anti-tumor cytotoxicity, antibody-dependent cellularcytotoxicity (ADCC), proliferation, and cytokine production (Trinchieri,Adv. Immunol. 47:187-376 (1989)). Also, IL-2-activated NK cells,frequently referred to as lymphokine-activated killer (LAK) cells,display a broader spectrum of reactivity against human and murine tumortarget cells. Thus, NK cells activated by IL-2 not only kill NKcell-sensitive tumor cells more efficiently, but also kill tumor cellsthat are insensitive to the constitutive cytotoxic activity mediated byNK cells.

[0007] Recent studies have also shown that IL-2, when combined withhistamine or serotonin, augments NK cell cytotoxicity in the presence ofmonocytes in vitro (Hellstrand et al., J. Immunol. 145(12):4365-4370(1990) and Hellstrand et al., Scand. J. Immunol. 32(2):183-192 (1990)).These studies suggest an interaction between monocytes and NK cells thatis subject to regulation by these biogenic amines (Hellstrand et al., J.Interferon Rsch. 12:199-206 (1992). These NK cell regulating mechanismsare thus believed to be of importance to the NK cell mediated responseto metastatic tumors in vivo.

[0008] Despite the beneficial effects obtained with IL-2 therapy inexperimental animals and despite the remarkable effects of IL-2 on thekilling activity of human NK cells in vitro, the results of the clinicaltrials of IL-2 in human cancer have, as yet, been disappointing. Only asmall fraction of patients with metastatic melanoma or renal cellcarcinoma show objective regression of tumor burden after treatment withvery high doses of IL-2 (Bukowski et al., J. Clin. Oncol. 7:477-485(1989); Whitehead et al., J. Natl. Cancer Inst. 83:1250-1253 (1991)). Inaddition, IL-2 produces severe side effects, including hypotension,fluid retention (“capillary leak syndrome”), fever, lethargy and nausea.

[0009] Other interleukins are also known to stimulate NK cell activity.For example, IL-12, also known as natural killer cell stimulatory factor(NKSF), is a recently discovered cytokine which has also been reportedto increase NK cell and cytotoxic T lymphocyte activity, T cellproliferation, and the production of interferon-γ. It has been found toenhance the spontaneous cytotoxic activity of peripheral bloodlymphocytes against a variety of tumor-derived target cell lines(Chehimi et al., J Exp. Med. 175:789-796 (1992)). IL-1 is anothercytokine known to enhance NK cell cytotoxicity.

[0010] The interferons consist of a family of secreted proteins withpotent antiproliferative and immunomodulatory activities. Theseimmunomodulatory effects include activation of macrophages, augmentationof cellular and humoral immune responses, and enhancement of NK-cellactivity. All three major subtypes of human interferon, i.e.,interferon-α (IFN-α), interferon-β (IFN-β) and interferon-γ (IFN-γ), areknown to enhance NK cell cytotoxicity. Interferon-α (IFN-α) is a majorregulatory factor for NK cells. It has been found to stimulate NK cells(Silva et al., J. Immunol. 125:479-484 (1980)) and augment NK cellcytotoxicity both in vitro and in vivo (Trinchieri, Adv. Immunol.47:187-376 (1989)). Although IFN-α has been shown to be effective withsome neoplasias, the overall results of therapy with high doses of IFN-αhave been disappointing. In addition, patients treated with IFN-α oftenhave acute toxic reactions including fever, chills, myalgias, anorexia,fatigue, headache, nausea and vomiting.

[0011] Other known stimulators of NK cell activity include certainflavonoids. The flavonoids are a group of low molecular weightpolyphenolic secondary plant metabolites. Flavone-8-acetic acid has beenfound to potently augment NK activity in the spleen, liver, lungs, andperitoneum (Wiltrout et al., J. Immunol. 140(9):3261-3265 (1988)).Xanthenone-4-acetic acid (XAA), an analog of FAA, and itsmethyl-substituted derivatives, have also been found to induce NKactivity in vitro (Ching et al., Eur. J. Cancer 27(1):79-83 (1991)).Clinical trials of FAA have been disappointing, however, due tonon-linear pharmokinetics, low dose potency and problems of drugprecipitation.

SUMMARY OF THE INVENTION

[0012] The present invention provides a novel method for the activationof NK cells and the prevention of inactivation of these cells bymonocytes, using a combination of a lymphokine or other NK cellactivator and a peroxide reducing or scavenging compound. The presentinvention is especially useful in the treatment of solid tumors andviral infection.

[0013] In accordance with one aspect of the present invention, there isprovided a method for providing activated natural killer cellscomprising the steps of administering to a population of cells whichincludes lymphocytes and monocytes, an effective amount of an NK cellactivating compound and a compound effective to inhibit the productionor release of intracellular hydrogen peroxide, provided that when saidNK cell activating compound is IL-2 or IFN-α, said compound effective toinhibit the production or release of intracellular hydrogen peroxide isnot histamine, an H₂ receptor agonist or serotonin.

[0014] In a preferred embodiment of the present invention, the compoundeffective to inhibit the production or release of intracellular hydrogenperoxide is histamine, an H₂ receptor agonist or serotonin, and the NKcell activating compound is a cytokine or a flavonoid. In anotherpreferred embodiment, the population of cells is located in vivo. Instill another preferred embodiment, the administration of said NK cellactivating compound and said compound effective to inhibit theproduction or release of intracellular hydrogen peroxide is performedsimultaneously. Alternatively, the administration of said NK cellactivating compound and said compound effective to inhibit theproduction or release of intracellular hydrogen peroxide is performedwithin 24 hours.

[0015] In another preferred embodiment of the present invention, the NKcell activating compound is a cytokine, which is administered in a doseof from about 1,000 to about 300,000 U/kg/day. In the preferredembodiment wherein the NK cell activating compound is a flavonoid, theflavonoid is administered in a dose of from about 1 to about 100,000mg/day. In still another preferred embodiment, the compound effective toinhibit the production or release of intracellular hydrogen peroxide isadministered in a dose of from about 0.1 to about 10 mg/day.

[0016] In accordance with another aspect of the present invention, thereis provided a method for providing activated natural killer cellscomprising the steps of administering to a population of cells whichincludes lymphocytes and monocytes, an effective amount of an NK cellactivating compound and administering a hydrogen peroxide scavenger. Ina preferred embodiment, the hydrogen peroxide scavenger catalyzes thedecomposition of hydrogen peroxide. In a preferred embodiment, thehydrogen peroxide scavenger is catalase, glutathione peroxidase, orascorbate peroxidase. In another preferred embodiment, the NK cellactivating compound is a cytokine or a flavonoid. In still anotherpreferred embodiment, the population of cells is located in vivo.

[0017] The administration of said NK cell activating compound and saidhydrogen peroxide scavenger is preferably performed simultaneously.Alternatively, the administration of said NK cell activating compoundand said hydrogen peroxide scavenger is performed within 24 hours. In apreferred embodiment, the NK cell activating compound is a cytokine, andthe cytokine is administered in a dose of from about 1,000 to about300,000 U/kg/day. In the preferred embodiment wherein the NK cellactivating compound is a flavonoid, the flavonoid is administered in adose of from about 1 to about 100,000 mg/day. Preferably, the hydrogenperoxide scavenger is administered in a dose of from about 0.1 to about10 mg/day.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018]FIG. 1 graphically depicts the activation of NK cells by catalaseand synergy with IL-2. Culture medium (control; open bars) or IL-2 (10U/ml; filled bars) was added to enriched human NK cells alone (FIG. 1A)or NK cells admixed with 30% monocytes (FIG. 1B) in the presence ofcatalase at the indicated final concentrations. The bars indicate NKcell-mediated killing of target tumor cells (cell lysis % “s.e.m. ofsextuplicate determination).

[0019]FIG. 2 graphically depicts the activation of NK cell-mediatedclearance of YAC-1 lymphoma cells in vivo by catalase. Seventy-fivethousand YAC-1 cells labeled with ⁵¹Cr were injected intravenously intomale or female 4-6-week-old Swiss Albino mice, together with vehicle(control; open bars) or catalase (100 U/kg; filled bars). Two hoursafter the inoculation of tumor cells, the mice were sacrificed bycervical dislocation. The results show retained radioactivity in lungtissue (% of radioactivity retained in lungs at t=0 after injection oflabeled tumor cells). Results from 4 separate experiments are shown.Each bar represents the mean “s.e.m. of 3-5 animals.

[0020]FIG. 3 shows the inhibition of IL-2-induced NK cell proliferationand cytotoxicity by monocytes and its reversal by histamine andcatalase. Culture medium (open bars), histamine (hatched bars), andcatalase (filled bars) were added to enriched NK cells (NK) or a mixtureof NK cells and monocytes (NK+MO). FIG. 3A shows NK cell proliferation,and FIG. 3B shows cytotoxicity against K562 target cells.

[0021]FIG. 4 illustrates the suppression of NK cell cytotoxicity byhydrogen peroxide and the role of myeloperoxidase. Open symbolsrepresent the cytotoxicity of cells treated with hydrogen peroxide.Filled symbols represent corresponding cells treated withmyeloperoxidase.

[0022]FIG. 5 illustrates the kinetics of monocyte-induced inhibition ofNK cell cytotoxicity. A mixture of NK cells and monocytes were treatedwith culture medium (open bars), histamine (hatched bars) or catalase(filled bars) at the indicated time points after the start of themicrotoxicity assay.

[0023]FIG. 6 shows that histamine inhibits the generation of hydrogenperoxide in monocytes. The luminol-enhanced chemiluminescence responseof monocytes treated with culture medium (solid line) or histamine(dotted line) is shown in FIG. 6A. FIG. 6B shows the response ofmonocytes treated with sodium azide (control, solid line), or histamineplus sodium azide (dotted line).

[0024]FIG. 7 shows that histamine H₂-type receptors transduce theeffects of histamine on the respiratory burst of monocytes. Monocyteswere treated with histamine plus ranitidine (open circles) or AH20239AA(filled circles).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0025] The present invention relates to a method for activating NK cellsin the presence of monocytes by using a hydrogen peroxide inhibiting orscavenging compound in combination with a cytokine or other NK cellactivator. The method of the present invention is useful, for example,as a method of inhibiting tumor growth and the formation of metastasesof malignant tumor cells, and in the treatment of viral infection.

[0026] In the monovalent pathway of oxygen reduction, superoxide anion(O₂) is produced first, followed by the formation of hydrogen peroxide(H₂O₂). Superoxide anion can react with hydrogen peroxide to formhydroxyl radical (OH). These reactive oxygen intermediates (ROI) areproduced by phagocytes such as monocytes and polymorphonuclearneutrophils (PMNs). Hydrogen peroxide produced by monocytes has beenfound to suppress NK cell mediated cytotoxicity. This NK cellcytotoxicity plays an important role in a host's defenses againstarising neoplasms and metastatic tumor cells in vivo. It has now beendiscovered that monocytes suppress NK cell cytotoxicity and that thismonocyte derived suppressive signal effectively down-regulates thecytotoxic and proliferative activities of NK cells. Suppression of NKcells has been found to be halted in the presence of the biogenic amineshistamine and serotonin (Hellstrand et al., J. Immunol. 145:4365-4370(1990)).

[0027] It is one of the surprising discoveries of the present inventionthat compounds which reduce the amount of hydrogen peroxide, whenadministered in combination with a cytokine or other compound known tostimulate NK cell activity, act to synergistically stimulate NK cellcytotoxicity in the presence of monocytes; thus, the administration ofscavengers of peroxide, or compounds which inhibit the production orrelease of intracellular peroxide, in combination with a cytokine orother NK cell activator, has been found effective in the treatment ofsolid tumors and viral infection.

[0028] Known scavengers of hydrogen peroxide include the enzymescatalase, glutathione peroxidase and ascorbate peroxidase. Compoundswhich inhibit the production or the release of intracellular peroxideare also effective in enhancing NK cell activity when administeredtogether with an NK cell activator. Such compounds include serotonin,histamine, and H₂ receptor agonists such as dimaprit.

[0029] The present invention therefore provides an effective method forpreventing the inactivation of NK cells and for activation these cells.It also provides a method for treatment of tumors and viral infection,through the administration of compounds which reduce the amount ofhydrogen peroxide, in combination with a cytokine or other compoundknown to stimulate NK cell activity. It is intended that the presentinvention cover the administration of the compounds listed and thosecompounds with similar activity, with the understanding that if thecytokine administered is IL-2 or IFN-α, the inhibitor is not histamine,an H₂ receptor agonist, or serotonin.

[0030] Administration of NK Cell Activator and Hydrogen PeroxideScavenger or Inhibitor

[0031] The administration of the cytokine or other compound known toenhance NK cell activity, together with the inhibiting or scavengingcompounds discussed above, can be by any of a number of methods wellknown to those of skill in the art. Such methods include the parenteraldelivery through intravenous, intraperitoneal, or intramuscularinjection. The NK cell activity enhancer and the hydrogen peroxidescavenger can be administered separately or as a single composition.When administered separately, it is contemplated that the NK cellactivity enhancer may be administered either first or last.

[0032] The compounds of the present invention may be administered inwater with or without a surfactant such as hydroxypropyl cellulose.Dispersions are also contemplated, such as those utilizing glycerol,liquid polyethylene glycols, and oils. Antimicrobial compounds may alsobe added to the preparations. Injectable preparations may includesterile aqueous solutions or dispersions and powders which may bediluted or suspended in a sterile environment prior to use. Carrierssuch as solvents or dispersion media contain water, ethanol polyols,vegetable oils and the like may also be added to the compounds of thepresent invention. Coatings such as lecithins and surfactants may beused to maintain the proper fluidity of the composition. Isotonic agentssuch as sugars or sodium chloride may be added, as well as productsintended to delay absorption of the active compounds such as aluminummonostearate and gelatin. Sterile injectable solutions are preparedaccording to methods well known to those of skill in the art and can befiltered prior to storage and/or use. Sterile powders may be vacuum orfreeze dried from a solution or suspension them. Sustained-releasepreparations and formulations are also contemplated by the presentinvention. Any material used in the composition of the present inventionshould be pharmaceutically acceptable and substantially non-toxic in theamounts employed.

[0033] All preparations may be provided in dosage unit forms for uniformdosage and ease of administration. Each dosage unit form contains apredetermined quantity of active ingredient calculated to produce adesired effect in association with an amount of pharmaceuticallyacceptable carrier.

[0034] Although in the Examples which follow the compounds areadministered as a single dose, it should be understood that thecompounds may be administered for prolonged periods of time. Typically,the treatment may be administered for periods up to about one week, andeven for periods longer than one month. In some instances, the treatmentmay be discontinued and then resumed at a later time. A daily dose maybe administered as a single dose, or it can be divided into severaldoses, especially if negative effects are observed. In addition, thecompounds of the present invention can be administered as a singlecomposition, or separately. If administered separately, the compoundsshould be given on the same day, such that the activation of NK cells bythe lymphokine or other compound is enhanced.

[0035] Preferred dosage range can be determined using techniques knownto those having ordinary skill in the art. IL-1, IL-2 or IL-12 can beadministered in an amount of from about 1,000 to about 300,000 U/kg/day;more preferable, the amount is from about 3,000 to about 100,000U/kg/day, and even more preferably, the amount is from about 5,000 toabout 20,000 U/kg/day.

[0036] IFN-α, IFN-β, and IFN-γ can also be administered in an amount offrom about 1,000 to about 300,000 U/kg/day; more preferable, the amountis from about 3,000 to about 100,000 U/kg/day, and even more preferably,the amount is from about 10,000 to about 50,000 U/kg/day.

[0037] Flavonoid compounds can be administered in an amount of fromabout 1 to about 100,000 mg/day; more preferable, the amount is fromabout 5 to about 10,000 mg/day, and even more preferably, the amount isfrom about 50 to about 1,000 mg/day.

[0038] Compounds which inhibit the release or formation of intracellularhydrogen peroxide, or scavengers of hydrogen peroxide, can beadministered in an amount of from about 0.1 to about 10 mg/day; morepreferable, the amount is from about 0.5 to about 8 mg/day, and evenmore preferably, the amount is from about 1 to about 5 mg/day. However,in each case, the dose depends on the activity of the administeredcompound. The foregoing doses are appropriate for histamine, catalaseand for H₂ receptor agonists. Appropriate doses for any particular hostcan be readily determined by empirical techniques well known to those ofordinary skill in the art.

[0039] The method of the present invention may be utilized alone or incombination with other anti-cancer therapies, as determined by thepractitioner.

[0040] Monocyte-Induced Inhibition of NK Cells: Reversal by Catalase andthe Role of Reactive Oxygen and Nitrogen Species

[0041] To investigate the effects of hydrogen peroxide scavengers on theactivation of NK cells by cytokines, we studied the effects of catalase,a heme containing enzyme that catabolizes hydrogen peroxide (H₂O₂) tooxygen and water, on human NK cell-mediated killing of tumor cells invitro and on NK cell function in mice in vivo. These experiments aredescribed below in Examples 1 and 2. The following examples are merelyillustrative of the present invention, and are not intended to limit theinvention in any way.

EXAMPLE 1

[0042] Using blood obtained from a healthy human blood donor, we studiedthe effects of catalase on NK cell-mediated killing of tumor cells. K562cells, from an NK sensitive erythroleukemic cell line, were used astarget cells in all experiments. Washed cells (10×10⁶ cells/ml) wereincubated with ⁵¹Cr (Amersham) at a concentration of 150 μCi/ml cellsuspension for 2-4 hours. After centrifugation and resuspension in cellculture medium, 10⁴ cells in 50 μl portions were added to the effectorcells in microplate wells.

[0043] In the first set of experiments, human NK cells alone (1.5×10⁵cells/well) were added to the K562 target cells. The combined cells werethen exposed to culture medium (control) or human recombinant IL-2(EuroCetus, Amsterdam, The Netherlands) at a final concentration of 10U/ml. The cells were then exposed to catalase (Boehringer-Mannheim) atconcentrations of 0 to 100 U/ml. These same conditions were repeatedusing human NK cells admixed with 30% human peripheral blood monocytes,recovered by centrifugal elutriation, added to the target K562 cells.

[0044] After incubation at 37° C. for 16 hours, supernatant fluids werecollected by a tissue collecting system (Amersham) and assayed forradioactivity in a gamma counter. Maximum ⁵¹Cr release was determined intarget cell cultures treated with Triton-X. NK cell cytotoxicity wascalculated as cell lysis % according to the following formula:${{cell}\quad {lysis}\quad \%} = {100 \times \frac{\begin{matrix}{{{experimental}\quad {release}} -} \\{{spontaneous}\quad {release}}\end{matrix}}{{{maximum}\quad {release}} - {{spontaneous}\quad {release}}}}$

[0045] The results of these studies are illustrated in FIG. 1. FIG. 1Ashows that catalase does not affect the level of NK-cell-mediatedkilling of tumor cells in the absence of monocytes, regardless ofwhether the NK cells are unactivated or activated by IL-2. FIG. 1B showsthat the anti-tumor activity of unactivated NK cells is suppressed bythe presence of monocytes. Further, catalase, at final concentrationsexceeding 10 U/ml, reverses this suppressive signal. FIG. 1A furthershows that IL-2 does not significantly activate NK-cell cytotoxicityagainst tumor cells in the presence of monocytes unless catalase ispresent.

[0046] Effects of Catalase and Other Scavengers

[0047] To study the relationship between the monocyte-derived inhibitorysignal and the respiratory burst activity of monocytes, we addedcatalase and various other scavengers of reactive oxygen metabolites toNK cells, alone or admixed with monocytes. We then measured thecytotoxicity of the NK cells against NK cell sensitive K562 target cellsas described above. The results of this testing are shown in Table I.TABLE I Effect of scavengers of reactive oxygen and nitrogen metaboliteson monocyte-induced inhibition of NK-cell cytotoxicity. exp. cell lysis%^(a) no. treatment conc. MO medium histamine 1 catalase 0 − 55 ″ 3 53 ″2 ″  20 U/ml − 60 ″ 3 52 ″ 4 ″ 0 + 19 ″ 2 57 ″ 1 ″  2.5 U/ml + 21 ″ 1 55″ 2 ″  5 U/ml + 34 ″ 2 55 ″ 2 ″  10 U/ml + 49 ″ 1 52 ″ 2 ″  20 U/ml + 57″ 2 50 ″ 4 SOD 200 U/ml − 52 ″ 2 54 ″ 2 ″ 200 U/ml + 1.3 ″ 2  54 ″ 2 2taurin 0 − 72 ″ 2 70 ″ 3 ″ 10⁻³M − 70 ″ 4 75 ″ 4 ″ 0 +  6 ″ 2 63 ″ 4 ″10⁻³M +  6 ″ 1 64 ″ 3 3 deferox 0 − 79 ″ 4 78 ″ 2 ″ 10⁻⁴M − 81 ″ 2 74 ″5 ″ 0 +  9 ″ 3 62 ″ 2 ″ 10⁻⁴M + 12 ″ 3 60 ″ 2 4 mannitol 0 − 77 ″ 4 69 ″2 ″ 3 × 10 ⁻⁴M − 73 ″ 3 68 ″ 3 ″ 0 + 19 ″ 2 64 ″ 4 ″ 3 × 10⁻⁴M + 20 ″ 167 ″ 2 5 L-NMMA 0 − 58 ″ 1 51 ″ 2 ″ 2 × 10⁻⁴M − 61 ″ 3 54 ″ 2 ″ 0 + 12 ″1 45 ″ 2 ″ 2 × 10⁻⁴M + 11 ″ 1 45 ″ 3

[0048] Catalase, which effectively degrades H₂O₂, had no effect on thecytotoxicity of NK cells in the absence of monocytes but was found tocompletely abrogate the monocyte-induced inhibition of baseline NK cellcytotoxicity. Catalase was effective at concentrations exceeding 5 U/ml.Histamine (histamine dihydrochloride; Sigma) at concentrations exceeding10⁻⁷ M abrogated the monocyte induced suppression of NK cells but wasineffective in the presence of catalase.

[0049] It was also discovered that superoxide dismutase (SOD), ascavenger of O₂ ⁻ did not alter the suppressive effect of monocytes onNK cells over a wide range of concentrations. Similarly, taurin, ascavenger of HOCl⁻, and scavengers of OH such as mannitol anddeferoxamine, were ineffective at reducing the suppressive effects ofmonocytes on NK cells.

[0050] Further, monocytes and macrophages produce reactive nitrogenintermediates of which nitric oxide (NO) is the ultimate effectormolecule. To study whether NO induction in monocytes contributed to NKcell inhibition, we used a NO synthetase inhibitor,N-monomethyl-L-arginine (L-NMMA). This compound, used at concentrationssufficient to inhibit induction of NO in monocytes, did not alter thesuppression of NK cell function by monocytes. These results are alsoshown in Table I.

[0051] Thus, we have concluded that NK cell-mediated cytotoxicity issuppressed by H₂O₂ produced by monocytes. This suppression of NKcell-mediated cytotoxicity induced by H₂O₂ is abrogated by the presenceof catalase or histamine. In addition, it was discovered that human NKcells do not respond to IL-2 unless the monocyte-derived H₂O₂ isscavenged by catalase or by some other scavenger.

[0052] In vivo Effects of Hydrogen Peroxide Scavengers

[0053] To study whether the regulatory effects on human NK cell functioninduced by catalase in vitro are of importance for NK cell-mediatedkilling of tumor cells in vivo, experiments were performed in whichcatalase was injected intravenously to mice shortly before intravenousinoculation of NK cell-sensitive tumor cells. These experiments aredescribed below in Example 2.

EXAMPLE 2

[0054] Seventy-five thousand ⁵¹Cr-labeled, NK cell-sensitive YAC-1 mouselymphoma cells were injected intravenously into male or female 4 to 6week old Swiss Albino mice, together with vehicle (control) or 100 U/kgcatalase. Two hours after inoculation with the tumor cells, the micewere sacrificed by cervical dislocation. The lungs were removed andplaced in test tubes in a gamma-counter, and the radioactivity in thelung tissue was measured. The radioactivity in lung tissue is an inversemeasure of NK cell-mediated killing of tumor cells in vivo (see Hanna etal., JNCI 65:801 (1980)), and is expressed as a percent of the ⁵¹Cr thatis retained in lungs immediately after inoculation of radiolabeled tumorcells.

[0055] The results of this testing are shown in FIG. 2. The data in FIG.2 represent four separate experiments. Each bar represents the retainedradioactivity in lung pairs (mean “s.e.m. of 3-5 animals). Consistently,it was found that treatment with catalase augmented the NK cell-mediatedkilling of YAC-1 lymphoma cells in vivo.

[0056] To determine whether the NK cell-mediated killing of tumor cellsis enhanced using other NK cell activators and other peroxidescavengers, the experiments described below are performed.

EXAMPLE 3

[0057] The experiment described in Example 1 is repeated using IL-12 asthe NK cell activator. Similar results are obtained.

EXAMPLE 4

[0058] The experiment described in Example 1 is repeated using IFN-α asthe NK cell activator. Similar results are obtained.

EXAMPLE 5

[0059] The experiment described in Example 1 is repeated using FAA asthe NK cell activator. Similar results are obtained.

EXAMPLE 6

[0060] The experiment described in Example 2 is repeated usingglutathione peroxidase as the hydrogen peroxide scavenger. Similarresults are obtained.

EXAMPLE 7

[0061] The experiment described in Example 2 is repeated using ascorbateperoxidase as the hydrogen peroxide scavenger. Similar results areobtained.

[0062] Inhibition of IL-2 Induced NK Cell Functions by Monocytes:Reversal by Catalase and Histamine

[0063] IL-2 activates NK cell mediated cytotoxicity and inducesproliferation of the resting population of NK cells. Elutriatedmonocytes effectively inhibit the IL-2 induced proliferation of enrichedNK cells as well as the activation of NK cell cytotoxicity. To show thathistamine, a compound we have discovered to suppress the generation ofH₂O₂ in monocytes, and catalase, a scavenger of H₂O₂, reverse themonocyte-induced inhibition, the following experiments were performed.

EXAMPLE 8

[0064] Cell culture medium (control), histamine (10⁻⁴ M), or catalase(20 U/ml), was added to either enriched NK cells alone or a mixture ofNK cells and monocytes in microplates (1.5×10⁵ cells/well). Each groupof cells then received 50 U/ml human recombinant IL-2 and were allowedto incubate for 48 hours. During the last 8 hours of incubation, cellswere pulsed with ³H-methyl-thymidine (specific activity 2 Ci/mole; NewEngland Nuclear Corp.; 1 μCi/2×10⁵ cells). Following incubation, thecells were collected on glass fiber filters with an automatic cellharvester and cell-incorporated ³H-methyl-thymidine was estimated byliquid scintillography.

[0065] The results are shown in FIG. 3A, which illustrates NK cellproliferation, as reflected by ³H-methyl-thymidine incorporation aftertreatment with IL-2. The bars represent cpm×10³ (proliferation)±s.e.m.of sextuplicates. The results show that monocytes inhibit theproliferation of NK cells induced by IL-2. Both histamine and catalaseeffectively reverse this monocyte-induced inhibition.

[0066] To show the inhibitory effect of monocytes on IL-2 induced NKcell cytotoxicity, and its reversal by histamine and catalase, thefollowing experiment was performed.

EXAMPLE 9

[0067] Cell culture medium (control), histamine (10⁻⁴ M), or catalase(20 U/ml), was added to either enriched NK cells alone or a mixture ofNK cells and monocytes (1.5×10⁵ cells/well). These mixtures were thenincubated with K562 target cells in sextuplicate in microplates in atotal volume of 200 μl and assayed for microcytotoxicity. Afterincubation at 37° C. for 16 hours in the presence of culture medium(control) or IL-2 (50 U/ml), supernatant fluids were collected andassayed for radioactivity as described above in connection withExample 1. The results are illustrated in FIG. 3B.

[0068]FIG. 3B shows the cytotoxicity of the respective cell mixturesagainst K562 target cells. The bars represent percent cell lysis±s.e.m.of sextuplicates. Again, it is clear from this data that monocytesinhibit the cytotoxicity of NK cells induced by IL-2. Both histamine andcatalase effectively reverse this monocyte-induced inhibition.

[0069] Reconstitution of Monocyte Induced Inhibition by H₂O₂

[0070] The finding that catalase, but not scavengers of O₂ or of OH;reversed the suppression of NK cells by monocytes suggests that H₂O₂, ormetabolites of this compound, is essential for expression of theinhibitory signal. We therefore studied whether hydrogen peroxide couldreconstitute the inhibitory effects of monocytes on NK cells.

EXAMPLE 10

[0071] Culture medium (control) or H₂O₂ at concentrations between 0-10μM was added to NK cell enriched lymphocytes for assay of cytotoxicityagainst ⁵¹Cr K562 target cells as described above. Addition of H₂O₂ toenriched NK cells effectively suppressed NK cell cytotoxicity. Theresults of this testing are shown in FIG. 4, which shows the cell lysis%±s.e.m. of sextuplicates. The ED₅₀ of H₂O₂ was approximately 2×10⁻⁶ M,as seen in FIG. 4.

[0072] It was also discovered that catalase (20 U/ml), but nothistamine, completely reversed the inhibition of NK cells induced byH₂O₂ (data not shown).

[0073] Role of MPO

[0074] To study whether H₂O₂ alone or its reactive metabolites mediatedthe inhibitory effect of exogenous H₂O₂ on NK cells, myeloperoxidase(MPO), a monocyte-derived enzyme that forms toxic hypohalous acids suchas HOCI from H₂O₂, and halides and OH⁻ from H₂H₂ and ferrous iron, wasadded to enriched NK cells, alone or together with H₂O₂. If radicalssuch as hypohalous acids contributed to the NK cell inhibitory signal,it was expected that MPO would potentiate the suppressive effect of H₂O₂on enriched NK cells. This testing is described below in Example 11.

EXAMPLE 11

[0075] MPO (100 U/ml) and H₂O₂ at concentrations between 0-10 μM wereadded to NK cell enriched lymphocytes for assay of cytotoxicity against⁵¹Cr K562 target cells as described above. The results of this testingare shown in FIG. 4, which shows the cell lysis %±s.e.m. ofsextuplicates.

[0076] MPO did not potentiate the suppressive effect of H₂O₂ on NKcells. It was found that addition of MPO slightly but significantlyscavenged H₂O₂ in these experiments.

[0077] These results, along with the finding the mannitol, taurin anddeferoxamine, all of which are scavengers of MPO catalyzed products, didnot affect the inhibition of NK cells by monocytes suggested that theinhibitory signal is independent of MPO activity.

[0078] Kinetics of the Monocyte Derived NK Cell Inhibitory Signal

[0079] To assess when the inhibitory signal is conveyed from monocytesto NK cells, experiments were performed in which catalase or histaminewere added to mixtures of monocytes and NK cells at various time pointsafter the beginning of the microcytotoxicity assay against K562 targetcells.

EXAMPLE 12

[0080] A mixture of enriched NK cells and monocytes were treated withculture medium (control), histamine (10⁻⁴ M), or catalase (20 U/ml) andassayed for microcytotoxicity against target K562 cells as describedabove in connection with Example 1. It was found that catalase andhistamine were effective in inhibiting the NK cell suppressive signalonly when these compounds were added within the first hour of incubationof monocytes with NK cells. The results of this testing are shown inFIG. 5. The data shown is cell lysis %±s.e.m. of sextuplicates.

[0081] In additional experiments, NK cell enriched lymphocytes werepretreated in petri dishes with culture medium (control), catalase orhistamine, in the concentrations indicated below in Table II, in thepresence or absence of monocytes. After 1 hour incubation, nonadherentlymphocytes were recovered, washed twice, and assayed for cytotoxicityas described above. We wished to determine whether the NK cellinhibitory signal was reversible by removal of monocytes and removal ofmonocyte-derived products. It was found that lymphocytes recovered frommonocyte/NK cell mixtures pretreated with catalase or histamine weremore cytotoxic against K562 target cells than control mixturespretreated with medium only. The results of this testing are shown belowin Table II. Data are cell lysis %±s.e.m. of sextuplicates and are theresults of 2 separate experiments. TABLE II Irreversible inhibition ofNK-cells by monocytes and H₂O₂. exp. no. pretreatment conc. MO celllysis %^(a) 1 medium − 66″3 histamine 10⁻⁵ M − 59″3 catalase 20 U/ml −67″2 medium + 14″1 histamine 10⁻⁵ M + 51″2 catalase 20 U/ml + 48″3 2medium − 59″3 H₂O₂ 1.5 × 10⁻⁶M − 26″2 ″   3 × 10⁻⁶M − 12″1 ″   6 × 10⁻⁶M−  2″1

[0082] These data show that the inhibition of NK cells is evoked withinthe first hour of incubation with monocytes and that the inhibition isnot reversible by removal of monocytes or monocyte derived factors. Toconfirm this finding, enriched NK cells were treated with H₂O₂ for 20minutes followed by extensive washing and assay for cytotoxicity.Pretreatment with H₂O₂ at micromolar concentrations was sufficient toeffectively inhibit NK cell cytotoxicity. The results of this testingare shown in Table II.

[0083] Histaminergic Regulation of the Respiratory Burst of Monocytes

[0084] Histamine has been reported to affect several functions ascribedto monocytes and macrophages, but effects of histamine on therespiratory burst of monocytes have remained unknown. To determine theseeffects, we first tested whether histamine could act as a scavenger ofH₂O₂ or its radical metabolites in a cell free system. We then studiedthe effects of histamine and H₂R-interactive compounds on therespiratory burst activity of monocytes. These experiments are describedin the following Examples.

EXAMPLE 13

[0085] Chemiluminescence (CL) of cells was recorded at 37° C. in a6-channel Biolumat LB 9505 (Berthold Co., Wildbad, Germany) using 4 mlpolypropylene tubes as described by Lock et al. Anal. Biochem. 173:450(1988). The reaction mixture contained 0.8 ml elutriated monocytes(5×10⁶ cells/ml). The tubes were allowed to equilibrate for 5 minutes at37° C. before formylmethionyl-leucyl-phenylalanine (fMLP; Sigma; 10⁻⁷ Mfinal concentration) and luminol (Sigma; 10⁻⁶ M) were added and lightemission recorded. Formylmethionyl-leucyl-phenylalanine was dissolved to10⁻² M in dimethyl sulfoxide and subsequently diluted in Krebs-Ringerphosphate buffer supplemented with glucose (10 mM), Ca²⁺ (1 mM), andMg²⁺ (1.5 mM). Luminol was dissolved in 0.1 mM NaOH to 5×10⁻² M andfurther diluted in Krebs-Ringer phosphate buffer.

[0086] The CL recorded with H₂O₂ and/or MPO (10 Fg/ml) or H₂O₂ andhorseradish peroxidase (HRP; Calbiochem, La Jolla, Calif.) was unchangedby histamine (10⁻⁴ M). Further, we used an assay system in which lysisof elutriated, ⁵¹Cr-labelled RBC was measured in microplates. Additionof H₂O₂ (5×10⁻⁵ M) to 10⁵ RBC induced lysis of approximately 50% of RBC.Histamine (10⁻⁴ M) did not alter the level of RBC killing induced byH₂O₂. It is therefore concluded that histamine is not a scavenger ofH₂O₂ or its radical metabolites.

EXAMPLE 14

[0087] In a second set of experiments, effects of histamine andH₂R-interactive compounds on the respiratory burst activity, as measuredby the luminol-enhanced CL response of enriched, elutriated monocytes,were studied. Monocytes were treated with culture medium (control),histamine (10⁻⁵ M), sodium azide (10⁻⁵ M), or histamine and sodium azideas described below. Emission of CL was recorded after addition of fMLPat time=0.

[0088] It was found that histamine effectively inhibited both the burstactivity of unstimulated monocytes and the induction of burst by fMLP.The results of this are shown in FIG. 6A. The inhibitory effect ofhistamine was dose dependent at final histamine concentrations of10⁻⁴−10⁻⁷ M.

[0089] To assess whether histamine acted by inhibiting the generation ofH₂O₂ or by reducing the availability of peroxidase, we next studied theeffects of histamine in monocytes treated with sodium azide to inhibitendogenous myeloperoxidase (MPO) and with exogenous, azide-insensitiveperoxidase (HRP) in excess. Histamine inhibited the fMLP induced CLresponse also in this type of assay, showing that histamine specificallyinhibits the formation of H₂O₂ in monocytes. The results of this testingare shown in FIG. 6B.

[0090] Dimaprit, (SK&F, Hertfordshire, England), a specific H₂R agonist,mimicked the effect of histamine on the respiratory burst of monocytes.In contrast, nor-dimaprit, (SK&F, Hertfordshire, England), an H₂Rinactive structural analog of dimaprit, was ineffective. A strikingdifference between histamine and dimaprit was that whereas histamineblocked respiratory burst activity within seconds, the effect ofdimaprit was not maximal until after 10-15 minutes of incubation (datanot shown).

[0091]FIG. 7 shows that the effects of histamine were entirely blockedby simultaneous treatment with the specific H₂R antagonist ranitidine(Glaxo). To exclude non-specific effects of ranitidine, we used aranitidine analog (AH20239AA; C₁₃H₂₂O₄; Glaxo) in which the thioether ofranitidine is replaced by an ether, thereby strongly reducing its H₂Rantagonist properties. In these experiments, monocytes were treated withhistamine (10⁻⁵ M) together with ranitidine or AH20239AA at finalconcentrations indicated in FIG. 7. All cells were treated with fMLP attime=0. Peak CL recorded in untreated monocytes (control) was 2.5×10⁷cpm.

[0092] The chemical control to ranitidine, AH20239AA, was more than100-fold less potent than ranitidine in antagonizing the effects ofhistamine on the suppression of NK cell function by monocytes, as wellas the inhibition of respiratory burst activity by histamine, as shownin FIG. 7. The effects of histamine on the respiratory burst activity ofmonocytes, therefore, are specifically transduced by H₂R.

[0093] Conclusion

[0094] We have discovered that hydrogen peroxide is a pivotal mediatorof monocyte-derived, NK cell suppressive signal. The inhibitoryeffective of hydrogen peroxide on NK cells was not catalyzed by theaddition of MPO, thus demonstrating that the MPO activity is notrequired to mediate NK cell inhibitory signals. Further, scavengers ofMPO catalyzed radicals do not affect the inhibition of NK cell functioninduced by monocytes.

[0095] It is clear from our results that histamine, serotonin, or otherH₂ receptor agonists, acting via monocyte H₂ receptors, inhibit thegeneration of reactive oxygen products by monocytes, and thereby inhibitthe NK cell suppressive signal. It is clear that scavengers of hydrogenperoxide also act to inhibit the NK cell suppressive signal.

[0096] We have thus shown that treatment with a combination of an NKcell activating cytokine or other compound and a hydrogen peroxidescavenger or inhibiting compound in the presence of monocytes preventsthe inactivation of NK cells and enhances NK cell cytotoxicity againsttumor cells. These are unexpectedly superior results, since undersimilar circumstances, NK cell activators alone had no such beneficialeffect. Of particular importance is that the potentiation of theanti-tumor effect of the NK cell activators induced by the concomitanttreatment with a peroxide scavenger or inhibiting compound permits areduction in the high doses of lymphokines which are used in cancertherapy. Advantageously, high dose treatments of lymphokines and theaccompanying serious side effects can be eliminated by the method of thepresent invention.

What is claimed is:
 1. A method for inhibiting tumor growth in a subjectsuffering from neoplastic disease comprising: identifying a subjectsuffering from neoplastic disease; administering to said subject aneffective amount of an NK cell activating cytokine or an NK cellactivating flavonoid, wherein said NK cell activating cytokine is notIL-2 or IFN-α; and administering a compound effective to inhibit theproduction or release of hydrogen peroxide selected from the groupconsisting of histamine, other H₂ receptor agonists, and serotonin. 2.The method of claim 1, wherein the administration of said NK cellactivating cytokine or said flavonoid and said compound effective toinhibit the production or release of intracellular hydrogen peroxide isperformed simultaneously.
 3. The method of claim 1, wherein theadministration of said compound effective to inhibit the production orrelease of intracellular hydrogen peroxide is performed within 24 hoursof the administration of said NK cell activating cytokine or flavonoid.4. The method of claim 1, wherein said cytokine is administered in adose of from about 1,000 to about 300,000 U/kg/day.
 5. The method ofclaim 1, wherein said flavonoid is administered in a dose of from about1 to about 100,000 mg/day.
 6. The method of claim 1, wherein saidhistamine, other H₂-receptor agonist or serotonin is administered in adose of from about 0.1 to about 10 mg/day.
 7. The method of claim 1,wherein said NK cell activating cytokine or flavonoid and said compoundeffective to inhibit the production or release of intracellular hydrogenperoxide are administered parenterally to said subject.
 8. A method forinhibiting tumor growth in a subject suffering from neoplastic diseasecomprising: identifying a subject suffering from neoplastic disease;administering to said subject an effective amount of an NK cellactivating cytokine or an NK cell activating flavonoid; andadministering an effective amount of a hydrogen peroxide scavenger. 9.The method of claim 8, wherein said hydrogen peroxide scavengercatalyzes the decomposition of hydrogen peroxide.
 10. The method ofclaim 9, wherein the compound is selected from the group consisting ofcatalase, glutathione peroxidase, and ascorbate peroxidase.
 11. Themethod of claim 8, wherein the administration of said NK cell activatingcytokine or said flavonoid and said hydrogen peroxide scavenger isperformed simultaneously.
 12. The method of claim 8, wherein theadministration of said NK cell activating cytokine or said flavonoid andsaid hydrogen peroxide scavenger is performed within 24 hours.
 13. Themethod of claim 8, wherein said cytokine is administered in a dose offrom about 1,000 to about 300,000 U/kg/day.
 14. The method of claim 8,wherein said flavonoid is administered in a dose of from about 1 toabout 100,000 mg/day.
 15. The method of claim 8, wherein said hydrogenperoxide scavenger is administered in a dose of from about 0.1 to about10 mg/day.
 16. The method of claim 8, wherein the administration of saidNK cell activating cytokine or said flavonoid and said hydrogen peroxidescavenger is performed parenterally.